Gene therapy offers the prospect of selectively introducing genes into cancer cells, leaving them susceptible to specific antitumor drugs. Current protocols to elicit tumor reduction utilize Herpes Simplex Virus type 1 (HSV) thymidine kinase (TK) with the prodrug ganciclovir (GCV), or the E. coli or yeast cytosine deaminase (CD) with the prodrug 5-fluorocytosine (5FC). While functional as suicide genes, a number of caveats restrict their full effectiveness. These include a poor Km or binding affinity for prodrugs and toxic side effects associated with the high prodrug doses necessary to elicit tumor response. We seek to identify the optimal suicide gene and prodrug combination for the safest and most effective cancer gene therapy. The specific aims of this project are to optimize three separate suicide gene systems [cytosine deaminase, guanylate kinase/TK (pathway engineering) and CD/TK (converging pathway engineering)] using mutagenesis strategies and to test the efficacy of enzyme variants in tumor cell lines and animal models. This research endeavors to overcome the kinetic limitations found in current suicide gene therapy strategies and will address and compare: 1) increasing production of activated prodrugs and the impact on tumor cell killing; 2) enhancing the bystander effect as it relates to increased cytotoxin production; 3) reducing prodrug doses for therapeutic efficacy to offset toxic side effects and; 4) augmenting synergy of the dual suicide gene approach (converging pathway engineering). Not only will the results from this project impact the choice of gene(s) used for cancer treatment but they also have broad application elsewhere including for graft versus host disease, restenosis, AIDS, in noninvasive tumor imaging, cell lineage ablation studies, in negative selection systems and selection against non-homologous recombination for the generation of transgenic mice.